Depletion of plasmacytoid dendritic cells

ABSTRACT

The present invention relates to antibodies targeted to BDCA2 that deplete plasmacytoid dendritic cells (pDC) and methods of using the antibodies to treat disorders associated with pDC.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/092,275, filed Apr. 6, 2016, which is a continuation of InternationalApplication No. PCT/US2014/070521, filed Dec. 16, 2014 which claims thebenefit of U.S. Provisional Application No. 61/916,322, filed Dec. 16,2013, the entire contents of each of which is fully incorporated hereinby reference.

STATEMENT OF FEDERAL SUPPORT

This invention was made with government support under Grant No. AI077454awarded by the National Institutes of Health. The government has certainrights in this invention.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R.§1.821, entitled 5470-680DV_ST25.txt, 34,708 bytes in size, generated onFeb. 13, 2017 and filed via EFS-Web, is provided in lieu of a papercopy. The Sequence Listing is incorporated herein by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

The present invention relates to antibodies targeted to BDCA2 thatdeplete plasmacytoid dendritic cells (pDC) and methods of using theantibodies to treat disorders associated with pDC.

BACKGROUND OF THE INVENTION

Plasmacytoid dendritic cells (pDC) are potent type I interferon (IFN-I)producing cells (Siegal et al., Science 284:1835 (1999)) and involved incontrolling various viral infections (Cervantes-Barragan et al., Proc.Natl. Acad. Sci. USA 109:3012 (2012); Takagi et al., Immunity 35:958(2011); Liu, Annu. Rev. Immunol. 23:275 (2005); Swiecki et al., Immunity33:955 (2010)). However, the contribution of pDC in humanimmunodeficiency virus-1 (HIV-1) infection and pathogenesis remainscontroversial. On one hand, pDC have been shown to inhibit HIV-1replication through IFN-1 production (Yonezawa et al., J. Virol. 77:3777(2003); Fong et al., J. Virol. 76:11033 (2002); Gurney et al., J.Immunol. 173:7269 (2004)). Moreover, the numerical and functionaldecline of pDC in HIV-1 infected patients correlates with opportunisticinfection independent of CD4′ T-cell counts (Siegal et al., J. Clin.Invest. 78:115 (1986); Feldman et al., Clin. Immunol. 101:201 (2001);Lichtner et al., Curr. HIV Res. 6:19 (2008)). On the other hand, pDC maycontribute to HIV immunopathogenesis. The sustained pDC activation andIFN-1 production in HIV-1 infected patients does not correlate withviral control but is predictive of disease progression (Buimovici-Kleinet al., Lancer 2:344 (1983); Buimovici-Klein et al., AIDS Res. 2:99-108(1986); Meier et al., Nature Medicine 15:955 (2009)). Additionally, pDCare activated during the acute phase of simian immunodeficiency virus(SIV) infection in both pathogenic Asian monkeys (Rhesus and cynomolgusmacaques) and non-pathogenic African monkeys (Sooty mangabeys andAfrican green monkeys). However, pDC activation is rapidly controlled inthe nonpathogenic SIV infection, whereas its activation and IFN-Iproduction are sustained during pathogenic infection in Asian monkey(Lederer et al., PLoS Pathogens 5:e1000296 (2009); Bosinger et al., J.Clin. Invest. 19:3556 (2009); Jacquelin et al., J. Clin. Invest.119:3544 (2009); Harris et al., J. Virol. 84:7886 (2010);Campillo-Gimenez et al., J. Virol. 84:1838 (2010)). Thus, theinteraction between HIV and pDCs is unclear.

The present invention addresses previous shortcomings in the art byproviding antibodies that deplete pDC in a subject and treat disordersassociated with pDC.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the identification ofantibodies that specifically bind to BDCA2 (blood dendritic cellantigen-2) and deplete pDC (e.g., reduces the number of pDC) whenadministered to a subject. The invention is based further on the use ofthese antibodies to deplete pDC in a subject and to treat disordersassociated with pDC in a subject.

Accordingly, in one aspect, the invention relates to methods ofdepleting pDC in a subject, comprising delivering to the subject anantibody or a fragment thereof that specifically binds to BDCA2 anddepletes pDC, thereby depleting pDC.

In another aspect, the invention relates to methods of treating adisorder associated with pDC in a subject, comprising delivering to thesubject an antibody or a fragment thereof that specifically binds toBDCA2 and depletes pDC, thereby treating the disorder.

In an additional aspect, the invention relates to the use of an antibodyor a fragment thereof that specifically binds to BDCA2 and depletes pDCin the preparation of a medicament for treating a disorder associatedwith pDC.

In another embodiment, the invention relates to the use of antibody or afragment thereof that specifically binds to BDCA2 and depletes pDC fortreating a disorder associated with pDC.

In a further aspect, the invention relates to antibodies or fragmentsthereof that specifically bind to BDCA2 and deplete pDC whenadministered to a subject.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1f show HIV-1 infection and immune-pathogenesis in humanizedmice infected with the pathogenic HIV-R3A isolate. (a) Viral RNA genomecopy numbers in plasma from mice inoculated with 1 ng p24/mouse of R3A(n=10). (b) Summary data for the percentages of HLA-DR+CD38+CD8 T cells(CD3+CD4−CD8+) in peripheral blood and spleen measured by FACS. (c)Summary data for the relative CD4+ T cells (CD3+CD8−CD4+) in total CD3+T cells. (d) Comparison of absolute CD4 T-cell, CD8 T-cell and huCD45+cell numbers in spleen from uninfected control mice (n=3) andR3A-infected mice (n=10). (e) The production of IFN-a2 in plasma fromuninfected (n=3) and infected (n=3) DKO-hu mice measured by luminex. (f)The relative level of Mx1 and TRIM22 gene expression in huCD45+ cell inspleen (n=3). All bars in dot graphs indicate median value. Error barsindicate standard deviations (SD). * and ** indicate p<0.05 and p<0.01,respectively.

FIG. 2 shows the kinetics of viremia in individual CCR5-tropicJR-CSF-infected DKO-hu mouse measured by quantitative real-time PCR(n=10).

FIGS. 3a-3c show CD8 T cell activation in acute R3A infection andchronic JR-CSF infection in DKO-hu mice. (a) Representative FACS plotsfor the percentages of HLA-DR+CD38+CD8 T cells in peripheral blood andspleen in R3A-infected mice at 3 weeks post-infection. (b)Representative FACS plots for the percentages of HLA-DR+CD38+CD8 T cellsin peripheral blood and spleen in JR-CSF-infected mice at 18 weekspost-infection. (c) Summarized data for supplementary FIG. 2b . mockn=4; JR-CSF n=10. All bars in dot graphs indicate median value. **indicate p<0.01.

FIGS. 4a-4c show CD4 T cell depletion in acute R3A infection and chronicJR-CSF infection in DKO-hu mice. (a) Representative FACS plots for thepercentages of CD4 T cells (CD8-CD4+) in peripheral blood and spleen inR3A-infected mice at 3 weeks post-infection. (b) Representative FACSplots for the percentages of CD4 T cells (CD8-CD4+) in peripheral bloodand spleen in R3A-infected mice at 18 weeks post-infection. (c)Summarized data for supplementary FIG. 3b . mock n=4; JR-CSF n=10. Allbars in dot graphs indicate median value. ** indicate p<0.01.

FIGS. 5a-5c show type I IFN response and HIV pathogenesis in R5-tropicJR-CSF-infected humanized mice terminated at 3 weeks post-infection. (a)Comparison of absolute CD4 T-cell, CD8 T-cell and human CD45+ cellnumbers in spleen from uninfected control mice (n=4) and JR-CSF-infectedmice (n=10). (b) The production of IFN-α2 in plasma from uninfected(n=3) and infected (n=3) humanized mice measured by luminex. (c) Therelative level of Mx1 and TRIM22 gene expression in human CD45+ cellsisolated from spleens. All bars in dot graphs indicate median value.Error bars indicate standard deviations (SD). * and ** indicate p<0.05and p<0.01, respectively.

FIGS. 6a-6b show depletion of pDC mediated by 15B in DKO-hu mice. (a-b)Humanized mice were treated with either 15B or isotype control (iso)antibody, pDC (CD4+CD123+) percentage of total human leukocytes (CD45+)were analyzed by FACS. (a) Representative FACS plots and summarized datashow relative pDC frequencies before and after antibody treatment inperipheral blood (n=7). (b) Representative FACS plots and summarizeddata show pDC depletion by 15B in mesenteric lymph nodes (mLN, isotypen=4; 15B n=5) and spleen (SP, isotype n=4; 15B n=5). All bars in dotgraphs indicate median value. Error bars indicate standard deviations(SD). * and ** indicate p<0.05 and p<0.01, respectively.

FIGS. 7a-7c show specific depletion of pDCs induced by 15B in differentlymphoid organs in DKO-hu mice. (a) Representative FACS plots showpercentages of CD3+CD19− cell and CD3-CD19+ cell in huCD45+ cells. (b-c)Summarized data for FIG. 7a . All bars in dot graphs indicate medianvalue.

FIGS. 8a-8b show specific depletion of pDCs induced by 15B in differentlymphoid organs in DKO-hu mice. (a) Representative FACS plots showpercentages of CD3-CD14+ cell in huCD45+ cells. (b) Summarized data forFIG. 8 a.

FIGS. 9a-9b show specific depletion of pDCs induced by 15B in differentlymphoid organs in DKO-hu mice. (a) Representative FACS plots showpercentages of CD3-CD11c+ cell in huCD45+ cells. (b) Summarized data forFIG. 9a . All bars in dot graphs indicate median value.

FIGS. 10a-10d show depletion of pDC abolishes IFN-I induction duringacute HIV-1 infection in DKO-hu mice. Humanized mice were treated witheither 15B or isotype control (iso) antibody. After pDC depletion,humanized mice were infected with HIV-R3A and terminated 8 days postinfection (dpi) for analysis. (a) Summarized data of pDC (CD4+CD123+)percentage in total human leukocytes (CD45+) analyzed by FACS. Mock,n=6; isotype+R3A, n=9; 15B+R3A, n=12. (b) Plasma IFNα2 of Mock, HIV-1infected and 15B treated mice were quantified by Luminex assay. Mock,n=3; isotype+R3A, n=5; 15B+R3A, n=5. (c-d) The mRNA levels of IFN-I andinterferon stimulated genes in purified human cells (CD45+) from mousespleen were measured by real-time PCR. Mock, n=3; isotype+R3A, n=5;15B+R3A, n=5. All bars in dot graphs indicate median value. Error barsindicate standard deviations (SD). * and ** indicate p<0.05 and p<0.01,respectively.

FIGS. 11a-11e show pre-depletion of pDC enhances HIV-1 replication.Humanized mice were infected with HIV-1 three days after pDC depletionand terminated at 8 dpi (R3A, a-b) or three weeks post infection(JR-CSF, c-e). (a) Plasma HIV-1 RNA levels (genome copy#× log 10/ml)were analyzed by real-time PCR. isotype+R3A, n=9; 15B+R3A, n=12. (b)Immunohistochemistry staining for p24 positive cells in spleens. (c)Plasma JR-CSF HIV-1 RNA levels (genome copy#× log 10/ml) were analyzedby real-time PCR at 3 weeks post infection. isotype+JR-CSF, n=12;15B+JR-CSF, n=12. (d) Representative FACS plots for p24 positive CD4 Tcells in spleens at 3 weeks post-infection. (e) Summarized data ofrelative p24+T cells. isotype+JR-CSF, n=7; 15B+JR-CSF, n=7. Bars in dotgraphs indicate median value. * indicates p<0.05.

FIGS. 12a-12d show elevated CD38+DR+CD8 T cells in pDC-depleted micewith elevated HIV-1 infection. (a) Representative FACS plots show CD38and HLA-DR expression on CD8 T cells induced by R3A infection inperipheral blood and spleen at 8 dpi. (b) Summarized data for FIG. 4a .(c) Representative FACS plots show CD8 T cell activation induced byJR-CSF infection at 3 weeks post-infection. (d) Summarized data for FIG.4c . mock, n=6; isotype+R3A, n=9; 15B+R3A, n=12. All bars in dot graphsindicate median value. * and ** indicate p<0.05 and p<0.01,respectively.

FIGS. 13a-13b shows the correlation between CD8 T cell activation inspleen and viral load. Correlations were analyzed with the Spearmannonparametric test. Isotype+R3A, n=9; 15B+R3A, n=12. Squared correlationcoefficients (r) and P values are shown.

FIGS. 14a-14e show pre-depletion of pDC reduces HIV-1immunopathogenesis. Humanized mice were infected with HIV-R3A three daysafter pDC depletion and terminated at 8 dpi. (a-c) Cell counts of humanT cells or total leukocytes in peripheral blood and spleen. (a) CD4 Tcell (CD3+CD8−) counts. (b) CD8 T cell (CD3+CD4-CD8+) counts. (c) Totalhuman CD45+ leukocyte counts. (d-e) Representative histograms andsummarized data show percentages of dead CD4 T cells, CD8 T cells andhuman CD45⁺ cells in spleens. Mock, n=6; isotype+R3A, n=9; 15B+R3A,n=12. All bars in dot graphs indicate median value. * and ** indicatep<0.05 and p<0.01, respectively.

FIGS. 15a-15c show pre-depletion of pDC reduced HIV-1 pathogenesis.Humanized mice were infected with HIV-1 three days after pDC depletionand terminated at 3 weeks post-infection. (a-c) Cell counts of human Tcells or total leukocytes in peripheral blood and spleen. (a) CD4 T cell(CD3+CD8−) counts. (b) CD8 T cell (CD3+CD4−CD8+) counts. (c) huCD45+leukocyte counts. Mock, n=4; isotype+JR-CSF, n=8; 15B3-JR-CSF, n=7. Allbars in dot graphs indicate median value. * indicates p<0.05.

FIGS. 16a-16g show depletion of pDC increases HIV-1 replication butreduces HIV-1 immunopathogenesis during chronic HIV-1 infection. HIV-1infected humanized mice were treated with 15B at 11 weeks post-infectionand terminated at 21 weeks post-infection (mock, n=6; JR-CSF+PBS, n=10;JR-CSF+15B, n=9). (a) Plasma HIV-1 RNA levels (genome copy#× log 10/ml)at each time point were analyzed by real-time PCR. (b) Summarized datashow percentages of HIV p24 positive CD4 T cells (CD3+CD8−) in spleens.(c-e) Cell counts of human T cells or total CD45 leukocytes inperipheral blood and spleens. (c) CD4 T cell (CD3+CD8−) counts. (d) CD8T cell (CD3+CD4-CD8+) counts. (e) Human CD45+ leukocyte counts. (f)Immunohistochemistry staining for human CD45+ cells in spleens. (g)Summarized data show percentages of dead CD4 T cells, CD8 T cells andhuman CD45⁺ cells in spleens (JR-CSF infection at termination, 21 wpi).Bars in dot graphs indicate median value. * and ** indicate p<0.05 andp<0.01, respectively.

FIGS. 17a-17f show depletion of pDC increases HIV-1 replication butreduces type I IFN response in chronic infection. HIV-1 infectedhumanized mice were started treatment with 15B at 11 weekspost-infection and terminated at 21 weeks post-infection. (a)Representative FACS histograms for FIG. 6b . (b) The production ofIFN-a2 in plasma from mock (n=4), JR-CSF+PBS (n=5) and JR-CSF+15B (n=5)at either 11 weeks post-infection (pre) or 21 weeks post infection(post), measured by Luminex. (c-d) The mRNA levels of IFN-1 andinterferon stimulated genes in purified human cells (CD45+) from mousespleen were measured by real-time PCR (n=5). (e-f) Relative ISGs geneexpression in purified human CD45+ cells (e) and CD8 T cells(CD3+CD4−CD8+) (f) from spleens at termination. All bars in dot graphsindicate median value. Error bars indicate standard deviations (SD). *and ** indicate p<0.05 and p<0.01, respectively.

FIG. 18 shows the nucleotide sequence (SEQ ID NO:1) and the amino acidsequence (SEQ ID NO:2) of the heavy chain of 15B.

FIG. 19 shows the nucleotide sequence (SEQ ID NO:3) and the amino acidsequence (SEQ ID NO:4) of the light chain of 15B.

FIG. 20 shows depletion of pDC mediated by 12B (also called 125) inDKO-hu mice.

FIG. 21 shows the nucleotide sequence (SEQ ID NO:5) and the amino acidsequence (SEQ ID NO:6) of the heavy chain of 12B.

FIG. 22 shows the nucleotide sequence (SEQ ID NO:7) and the amino acidsequence (SEQ ID NO:8) of the light chain of 12B.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in more detail withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a complex comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

Nucleotide sequences are presented herein by single strand only, in the5′ to 3′ direction, from left to right, unless specifically indicatedotherwise. Nucleotides and amino acids are represented herein in themanner recommended by the IUPAC-IUB Biochemical Nomenclature Commission,or (for amino acids) by either the one-letter code, or the three lettercode, both in accordance with 37 C.F.R. §1.822 and established usage.

Except as otherwise indicated, standard methods known to those skilledin the art may be used for cloning genes, amplifying and detectingnucleic acids, and the like. Such techniques are known to those skilledin the art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual 2nd Ed. (Cold Spring Harbor, N.Y., 1989); Ausubel et al. CurrentProtocols in Molecular Biology (Green Publishing Associates, Inc. andJohn Wiley & Sons, Inc., New York).

All publications, patent applications, patents, patent publications andother references cited herein are incorporated by reference in theirentireties for the teachings relevant to the sentence and/or paragraphin which the reference is presented.

I. Definitions

As used in the description of the invention and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

The term “about,” as used herein when referring to a measurable valuesuch as an amount of polypeptide, dose, time, temperature, enzymaticactivity or other biological activity and the like, is meant toencompass variations of 20%, ±10%, +5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

The transitional phrase “consisting essentially of” means that the scopeof a claim is to be interpreted to encompass the specified materials orsteps recited in the claim, “and those that do not materially affect thebasic and novel characteristic(s)” of the claimed invention. See, In reHerz, 537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (emphasis inthe original); see also MPEP §2111.03.

The term “consists essentially of” (and grammatical variants), asapplied to a polynucleotide or polypeptide sequence of this invention,means a polynucleotide or polypeptide that consists of both the recitedsequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5′and/or 3′ or N-terminal and/or C-terminal ends of the recited sequencesuch that the function of the polynucleotide or polypeptide is notmaterially altered. The total of ten or less additional nucleotides oramino acids includes the total number of additional nucleotides or aminoacids on both ends added together. The term “materially altered,” asapplied to polynucleotides of the invention, refers to an increase ordecrease in ability to express the encoded polypeptide of at least about50% or more as compared to the expression level of a polynucleotideconsisting of the recited sequence. The term “materially altered,” asapplied to polypeptides of the invention, refers to an increase ordecrease in epitope binding activity of at least about 50% or more ascompared to the activity of a polypeptide consisting of the recitedsequence.

An “effective” amount as used herein is an amount that provides adesired effect.

A “therapeutically effective” amount as used herein is an amount thatprovides some improvement or benefit to the subject. Alternativelystated, a “therapeutically effective” amount is an amount that willprovide some alleviation, mitigation, or decrease in at least oneclinical symptom in the subject (e.g., in the case of HIV infection,reduction in viral load or increase in immune cells). Those skilled inthe art will appreciate that the therapeutic effects need not becomplete or curative, as long as some benefit is provided to thesubject.

By the terms “treat,” “treating,” or “treatment of,” it is intended thatthe severity of the subject's condition is reduced or at least partiallyimproved or modified and that some alleviation, mitigation or decreasein at least one clinical symptom is achieved.

The term “deplete,” as used herein with respect to pDC, refers to ameasurable decrease in the number of pDC in a subject or in a sample.The reduction can be at least about 10%, e.g., at least about 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. Incertain embodiments, the term refers to a decrease in the number of pDCin a subject or in a sample to an amount below detectable limits.

The phrase “disorder associated with pDC,” as used herein, refers to anydisease, disorder, or condition in which pDC play a role in a cause,side effect, symptom, or other aspect in the disease, disorder, orcondition. Examples of such disorders include, without limitation,infectious diseases, autoimmune disorders, and cancer.

The term “infectious diseases,” as used herein, refers to any diseaseassociated with infection by an infectious agent. Examples of infectiousagents include, without limitation, viruses and microorganisms. Virusesinclude, without limitation, Hepadnaviridae including hepatitis A, B, C,D, E, F, G, etc.; Flaviviridae including human hepatitis C virus (HCV),yellow fever virus and dengue viruses; Retroviridae including humanimmunodeficiency viruses (HIV) and human T lymphotropic viruses (HTLV1and HTLV2); Herpesviridae including herpes simplex viruses (HSV-1 andHSV-2), Epstein Barr virus (EBV), cytomegalovirus, varicella-zostervirus (VZV), human herpes virus 6 (HHV-6) human herpes virus 8 (HHV-8),and herpes B virus; Papovaviridae including human papilloma viruses;Rhabdoviridae including rabies virus; Paramyxoviridae includingrespiratory syncytial virus; Reoviridae including rotaviruses;Bunyaviridae including hantaviruses; Filoviridae including Ebola virus;Adenoviridae; Parvoviridae including parvovirus B-19; Arenaviridaeincluding Lassa virus; Orthomyxoviridae including influenza viruses;Poxviridae including Orf virus, molluscum contageosum virus, smallpoxvirus and Monkey pox virus; Togaviridae including Venezuelan equineencephalitis virus; Coronaviridae including corona viruses such as thesevere acute respiratory syndrome (SARS) virus; and Picornaviridaeincluding polioviruses; rhinoviruses; orbiviruses; picodnaviruses;encephalomyocarditis virus (EMV); Parainfluenza viruses, adenoviruses,Coxsackieviruses, Echoviruses, Rubeola virus, Rubella virus, humanpapillomaviruses, Canine distemper virus, Canine contagious hepatitisvirus, Feline calicivirus, Feline rhinotracheitis virus, TGE virus(swine), Foot and mouth disease virus, simian virus 5, humanparainfluenza virus type 2, human metapneuomovirus, enteroviruses, andany other pathogenic virus now known or later identified (see, e.g.,Fundamental Virology, Fields et al., Eds., 3^(rd) ed., Lippincott-Raven,New York, 1996, the entire contents of which are incorporated byreference herein for the teachings of pathogenic viruses).

Pathogenic microorganisms include, but are not limited to, Rickettsia,Chlamydia, Mycobacteria, Clostridia, Corynebacteria, Mycoplasma,Ureaplasma, Legionella, Shigella, Salmonella, pathogenic Escherichiacoli species, Bordatella, Neisseria, Treponema, Bacillus, Haemophilus,Moraxella, Vibrio, Staphylococcus spp., Streptococcus spp.,Campylobacter spp., Borrelia spp., Leptospira spp., Erlichia spp.,Klebsiella spp., Pseudomonas spp., Helicobacter spp., and any otherpathogenic microorganism now known or later identified (see, e.g.,Microbiology, Davis et al, Eds., 4^(th) ed., Lippincott, New York, 1990,the entire contents of which are incorporated herein by reference forthe teachings of pathogenic microorganisms). Specific examples ofmicroorganisms include, but are not limited to, Helicobacter pylori,Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum,Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pyogenes,Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis,Neisseria meningitidis, Neisseria gonorrhoeae, Treponema pallidum,Bacillus anthracis, Salmonella typhi, Vibrio cholera, Pasteurella pestis(Yersinia pestis), Pseudomonas aeruginosa, Campylobacter jejuni,Clostridium difficile, Clostridium botulinum, Mycobacteriumtuberculosis, Borrelia burgdorferi, Haemophilus ducreyi, Corynebacteriumdiphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetellabronchiseptica, Haemophilus influenza, and enterotoxic Escherichia coli.

The term “autoimmune disorders,” as used herein, refers to any disorderassociated with an autoimmune reaction. Examples include, withoutlimitation, multiple sclerosis, Crohn's disease, ulcerative colitis,lupus, inflammatory bowel syndrome, and irritable bowel syndrome.

The term “cancer,” as used herein, refers to any benign or malignantabnormal growth of cells. Examples include, without limitation, breastcancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, coloncancer, melanoma, malignant melanoma, ovarian cancer, brain cancer,primary brain carcinoma, head-neck cancer, glioma, glioblastoma, livercancer, bladder cancer, non-small cell lung cancer, head or neckcarcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma,small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicularcarcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma,colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroidcarcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenalcarcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortexcarcinoma, malignant pancreatic insulinoma, malignant carcinoidcarcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia,cervical hyperplasia, leukemia, acute lymphocytic leukemia, chroniclymphocytic leukemia, acute myelogenous leukemia, chronic myelogenousleukemia, chronic granulocytic leukemia, acute granulocytic leukemia,hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma,polycythemia vera, essential thrombocytosis, Hodgkin's disease,non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primarymacroglobulinemia, and retinoblastoma. In some embodiments, the canceris selected from the group of tumor-forming cancers.

As used herein, “nucleic acid,” “nucleotide sequence,” and“polynucleotide” are used interchangeably and encompass both RNA andDNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemicallysynthesized) DNA or RNA and chimeras of RNA and DNA. The termpolynucleotide, nucleotide sequence, or nucleic acid refers to a chainof nucleotides without regard to length of the chain.

The term “isolated” can refer to a nucleic acid, nucleotide sequence orpolypeptide that is substantially free of cellular material, viralmaterial, and/or culture medium (when produced by recombinant DNAtechniques), or chemical precursors or other chemicals (when chemicallysynthesized). Moreover, an “isolated fragment” is a fragment of anucleic acid, nucleotide sequence or polypeptide that is not naturallyoccurring as a fragment and would not be found in the natural state.“Isolated” does not mean that the preparation is technically pure(homogeneous), but it is sufficiently pure to provide the polypeptide ornucleic acid in a form in which it can be used for the intended purpose.

The term “fragment,” as applied to a polynucleotide, will be understoodto mean a nucleotide sequence of reduced length relative to a referencenucleic acid or nucleotide sequence and comprising, consistingessentially of, and/or consisting of a nucleotide sequence of contiguousnucleotides identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99%identical) to the reference nucleic acid or nucleotide sequence. Such anucleic acid fragment according to the invention may be, whereappropriate, included in a larger polynucleotide of which it is aconstituent. In some embodiments, such fragments can comprise, consistessentially of, and/or consist of oligonucleotides having a length of atleast about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, or more consecutive nucleotides of a nucleic acid or nucleotidesequence according to the invention.

The term “fragment,” as applied to a polypeptide, will be understood tomean an amino acid sequence of reduced length relative to a referencepolypeptide or amino acid sequence and comprising, consistingessentially of, and/or consisting of an amino acid sequence ofcontiguous amino acids identical or almost identical (e.g., 90%, 92%,95%, 98%, 99% identical) to the reference polypeptide or amino acidsequence. Such a polypeptide fragment according to the invention may be,where appropriate, included in a larger polypeptide of which it is aconstituent. In some embodiments, such fragments can comprise, consistessentially of, and/or consist of peptides having a length of at leastabout 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, or more consecutive amino acids of a polypeptide or amino acidsequence according to the invention.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably and encompass both peptides and proteins, unlessindicated otherwise.

A “fusion protein” is a polypeptide produced when two heterologousnucleotide sequences or fragments thereof coding for two (or more)different polypeptides not found fused together in nature are fusedtogether in the correct translational reading frame. Illustrative fusionpolypeptides include fusions of a polypeptide of the invention (or afragment thereof) to all or a portion of glutathione-S-transferase,maltose-binding protein, or a reporter protein (e.g., Green FluorescentProtein, β-glucuronidase, β-galactosidase, luciferase, etc.),hemagglutinin, c-myc, FLAG epitope, etc.

As used herein, a “functional” polypeptide or “functional fragment” isone that substantially retains at least one biological activity normallyassociated with that polypeptide (e.g., target protein binding). Inparticular embodiments, the “functional” polypeptide or “functionalfragment” substantially retains all of the activities possessed by theunmodified peptide. By “substantially retains” biological activity, itis meant that the polypeptide retains at least about 20%, 30%, 40%, 50%,60%, 75%, 85%, 90%, 95%, 97%, 98%, 991%, or more, of the biologicalactivity of the native polypeptide (and can even have a higher level ofactivity than the native polypeptide). A “non-functional” polypeptide isone that exhibits little or essentially no detectable biologicalactivity normally associated with the polypeptide (e.g., at most, onlyan insignificant amount, e.g., less than about 10% or even 5%).Biological activities such as protein binding can be measured usingassays that are well known in the art and as described herein.

II. ANTIBODIES AND COMPOSITIONS

The inventors have identified and characterized antibodies thatspecifically bind to BDCA2 and deplete pDC. Such antibodies canadvantageously be used to deplete pDC in a subject, e.g., for researchor therapeutic purposes. Such antibodies can be used to treat disordersassociated with pDC. Accordingly, one aspect of the invention relates toantibodies or fragments thereof that specifically bind to BDCA2 anddepletes pDC when administered to a subject.

The term “antibody” or “antibodies” as used herein refers to all typesof immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodycan be monoclonal or polyclonal and can be of any species of origin,including (for example) mouse, rat, rabbit, horse, goat, sheep, camel,or human, or can be a chimeric antibody. See, e.g., Walker et al.,Molec. Immunol. 26:403 (1989). The antibodies can be recombinantmonoclonal antibodies produced according to the methods disclosed inU.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies canalso be chemically constructed according to the method disclosed in U.S.Pat. No. 4,676,980.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, Fab′, F(ab′)₂, and Fv fragments; domainantibodies, diabodies; vaccibodies, linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. Such fragments can be produced by known techniques. Forexample, F(ab′)₂ fragments can be produced by pepsin digestion of theantibody molecule, and Fab fragments can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fabexpression libraries can be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse et al., Science 254:1275 (1989)).

Antibodies of the invention may be altered or mutated for compatibilitywith species other than the species in which the antibody was produced.For example, antibodies may be humanized or camelized. Humanized formsof non-human (e.g., murine) antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementarity determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework (FR) regions (i.e., the sequences between the CDR regions)are those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522 (1986); Riechmann et al.,Nature, 332:323 (1988); and Presta, Curr. Op. Struct. Biol. 2:593(1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canessentially be performed following the method of Winter and co-workers(Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323(1988); Verhoeyen et al., Science 239:1534 (1988)), by substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567), wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues (e.g., all of theCDRs or a portion thereof) and possibly some FR residues are substitutedby residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)).The techniques of Cole et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol. 147:86 (1991)). Similarly, human antibodies can be madeby introducing human immunoglobulin loci into transgenic animals, e.g.,mice in which the endogenous immunoglobulin genes have been partially orcompletely inactivated. Upon challenge, human antibody production isobserved, which closely resembles that seen in humans in all respects,including gene rearrangement, assembly, and antibody repertoire. Thisapproach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in thefollowing scientific publications: Marks et al., Bio/Technology 10:779(1992); Lonberg et al., Nature 368:856 (1994); Morrison, Nature 368:812(1994); Fishwild et al., Nature Biotechnol. 14:845 (1996); Neuberger,Nature Biotechnol. 14:826 (1996); Lonberg and Huszar, Intern. Rev.Immunol. 13:65 (1995).

Polyclonal antibodies used to carry out the present invention can beproduced by immunizing a suitable animal (e.g., rabbit, goat, etc.) withan antigen to which a monoclonal antibody to the target binds,collecting immune serum from the animal, and separating the polyclonalantibodies from the immune serum, in accordance with known procedures.The polynucleotide sequence and polypeptide sequence of BDCA2 is knownin the art and can be found in sequence databases such as GenBank.Examples of sequences include the human BDCA2 polypeptide sequence(Accession No. Q8WTT0) and polynucleotide sequence (Accession No.AF293615), incorporated herein by reference in their entirety.

Monoclonal antibodies used to carry out the present invention can beproduced in a hybridoma cell line according to the technique of Kohlerand Milstein, Nature 265:495 (1975). For example, a solution containingthe appropriate antigen can be injected into a mouse and, after asufficient time, the mouse sacrificed and spleen cells obtained. Thespleen cells are then immortalized by fusing them with myeloma cells orwith lymphoma cells, typically in the presence of polyethylene glycol,to produce hybridoma cells. The hybridoma cells are then grown in asuitable medium and the supernatant screened for monoclonal antibodieshaving the desired specificity. Monoclonal Fab fragments can be producedin E. coli by recombinant techniques known to those skilled in the art.See, e.g., Huse, Science 246:1275 (1989).

Antibodies specific to the target polypeptide can also be obtained byphage display techniques known in the art.

Various immunoassays can be used for screening to identify antibodieshaving the desired specificity for BDCA2. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificity are well known inthe art. Such immunoassays typically involve the measurement of complexformation between an antigen and its specific antibody (e.g.,antigen/antibody complex formation). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on the polypeptides or peptides of thisinvention can be used as well as a competitive binding assay.

Antibodies can be conjugated to a solid support (e.g., beads, plates,slides or wells formed from materials such as latex or polystyrene) inaccordance with known techniques. Antibodies can likewise be conjugatedto detectable groups such as radiolabels (e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzymelabels (e.g., horseradish peroxidase, alkaline phosphatase), andfluorescence labels (e.g., fluorescein) in accordance with knowntechniques. Determination of the formation of an antibody/antigencomplex in the methods of this invention can be by detection of, forexample, precipitation, agglutination, flocculation, radioactivity,color development or change, fluorescence, luminescence, etc., as iswell known in the art.

In one embodiment, the antibody is an antibody or a fragment thereof(e.g., a monoclonal antibody) that specifically binds to BDCA2. Theantibody may bind to a specific epitope on BDCA2.

In one embodiment, the antibody is a monoclonal antibody produced byhybridoma cell line 15B (ATCC Deposit No. ______). In a furtherembodiment, the antibody is a monoclonal antibody or a fragment thereofthat competes for binding to the same epitope specifically bound by themonoclonal antibody produced by hybridoma cell line 15B. In anotherembodiment, the antibody is a monoclonal antibody or a fragment thereofthat specifically binds to the same epitope specifically bound by themonoclonal antibody produced by hybridoma cell line 15B. The epitopebound by the antibody produced by hybridoma cell line 15B comprises,consists essentially of, or consists of the amino acid sequenceIQNLKRNSSYFLGLSDPGGR (SEQ ID NO:9) or a fragment thereof of at least 5contiguous amino acids, e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 or more contiguous amino acids.

In certain embodiments, the monoclonal antibody or a fragment thereof isa chimeric antibody or a humanized antibody. In additional embodiments,the chimeric or humanized antibody comprises at least a portion of theCDRs of the monoclonal antibody produced by hybridoma cell line 15B. Asused herein, a “portion” of a CDR is defined as one or more of the threeloops from each of the light and heavy chain that make up the CDRs(e.g., from 1-6 of the CDRs) or one or more portions of a loopcomprising, consisting essentially of, or consisting of at least threecontiguous amino acids. For example, the chimeric or humanized antibodymay comprise 1, 2, 3, 4, 5, or 6 CDR loops, portions of 1, 2, 3, 4, 5,or 6 CDR loops, or a mixture thereof, in any combination.

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:2or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a heavy chain variable region comprising anamino acid sequence encoded by the nucleotide sequence of SEQ ID NO:1 ora sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In some embodiments, the antibody orfragment thereof comprises a heavy chain variable region comprising atleast 50 contiguous amino acids of the amino acid sequence of SEQ IDNO:2 or a sequence at least 90% identical thereto, e.g., at least 100,150, or 200 or more contiguous amino acids.

In one embodiment, the antibody or a fragment thereof comprises a lightchain variable region comprising the amino acid sequence of SEQ ID NO:4or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a light chain variable region comprising anamino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3 ora sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In some embodiments, the antibody orfragment thereof comprises a light chain variable region comprising atleast 50 contiguous amino acids of the amino acid sequence of SEQ IDNO:4 or a sequence at least 90% identical thereto, e.g., at least 100,150, or 200 or more contiguous amino acids.

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising the amino acid sequence of SEQ II) NO:2or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto, or encoded by the nucleotide sequence ofSEQ ID NO:1 or a sequence at least 90% identical thereto, e.g., at least95, 96, 97, 98, or 99% identical thereto, and a light chain variableregion comprising the amino acid sequence of SEQ ID NO:4 or a sequenceat least 90% identical thereto, e.g., at least 95, 96, 97, 98, or 99%identical thereto, or encoded by the nucleotide sequence of SEQ ID NO:3or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In some embodiments, the antibody orfragment thereof comprises a heavy chain variable region comprising atleast 50 contiguous amino acids of the amino acid sequence of SEQ IDNO:2 or a sequence at least 90% identical thereto, e.g., at least 100,150, or 200 or more contiguous amino acids, and a light chain variableregion comprising at least 50 contiguous amino acids of the amino acidsequence of SEQ ID NO:4 or a sequence at least 90% identical thereto,e.g., at least 100, 150, or 200 or more contiguous amino acids.

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising at least one CDR (e.g., 1, 2, or 3) ora portion thereof from the amino acid sequence of SEQ ID NO:2 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a heavy chain variable region comprising atleast one CDR (e.g., 1, 2, or 3) or a portion thereof from an amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:1 or a sequenceat least 90% identical thereto, e.g., at least 95, 96, 97, 98, or 99%identical thereto. One of skill in the art understands that the CDRsplay an important role in binding specificity and that sequencesubstitutions (e.g., for humanization of a mouse antibody) arepreferably made outside of the CDRs and that minimal changes are madewithin the CDRs. Thus, in some embodiments, sequences that are at least90% identical to the disclosed sequences comprise no changes or only aminimal number of changes to the CDRs.

In one embodiment, the antibody or a fragment thereof comprises a lightchain variable region comprising at least one CDR (e.g., 1, 2, or 3) ora portion thereof from the amino acid sequence of SEQ ID NO:4 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a light chain variable region comprising atleast one CDR (e.g., 1, 2, or 3) or a portion thereof from an amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:3 or a sequenceat least 90% identical thereto, e.g., at least 95, 96, 97, 98, or 99%identical thereto.

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising at least one CDR (e.g., 1, 2, or 3)from the amino acid sequence of SEQ ID NO:2 or a sequence at least 90%identical thereto, e.g., at least 95, 96, 97, 98, or 99% identicalthereto, or encoded by the nucleotide sequence of SEQ ID NO:1 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto, and a light chain variable region comprisingat least one CDR (e.g., 1, 2, or 3) from the amino acid sequence of SEQID NO:4 or a sequence at least 90% identical thereto, e.g., at least 95,96, 97, 98, or 99% identical thereto, or encoded by the nucleotidesequence of SEQ ID NO:3 or a sequence at least 90% identical thereto,e.g., at least 95, 96, 97, 98, or 99% identical thereto.

In one embodiment, the antibody is a monoclonal antibody produced byhybridoma cell line 12B (previously called 125) (ATCC Deposit No.______). In a further embodiment, the antibody is a monoclonal antibodyor a fragment thereof that competes for binding to the same epitopespecifically bound by the monoclonal antibody produced by hybridoma cellline 12B. In another embodiment, the antibody is a monoclonal antibodyor a fragment thereof that specifically binds to the same epitopespecifically bound by the monoclonal antibody produced by hybridoma cellline 12B. In certain embodiments, the monoclonal antibody or a fragmentthereof is a chimeric antibody or a humanized antibody. In additionalembodiments, the chimeric or humanized antibody comprises at least aportion of the CDRs of the monoclonal antibody produced by hybridomacell line 12B.

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:6or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a heavy chain variable region comprising anamino acid sequence encoded by the nucleotide sequence of SEQ ID NO:5 ora sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In some embodiments, the antibody orfragment thereof comprises a heavy chain variable region comprising atleast 50 contiguous amino acids of the amino acid sequence of SEQ IDNO:6 or a sequence at least 90% identical thereto, e.g., at least 100,150, or 200 or more contiguous amino acids.

In one embodiment, the antibody or a fragment thereof comprises a lightchain variable region comprising the amino acid sequence of SEQ ID NO:8or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a light chain variable region comprising anamino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7 ora sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In some embodiments, the antibody orfragment thereof comprises a light chain variable region comprising atleast 50 contiguous amino acids of the amino acid sequence of SEQ IDNO:8 or a sequence at least 90% identical thereto, e.g., at least 100,150, or 200 or more contiguous amino acids.

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:6or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto, or encoded by the nucleotide sequence ofSEQ ID NO:5 or a sequence at least 90% identical thereto, e.g., at least95, 96, 97, 98, or 99% identical thereto, and a light chain variableregion comprising the amino acid sequence of SEQ ID NO:8 or a sequenceat least 90% identical thereto, e.g., at least 95, 96, 97, 98, or 99%identical thereto, or encoded by the nucleotide sequence of SEQ ID NO:7or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In some embodiments, the antibody orfragment thereof comprises a heavy chain variable region comprising atleast 50 contiguous amino acids of the amino acid sequence of SEQ IDNO:6 or a sequence at least 90% identical thereto, e.g., at least 100,150, or 200 or more contiguous amino acids, and a light chain variableregion comprising at least 50 contiguous amino acids of the amino acidsequence of SEQ ID NO:8 or a sequence at least 90% identical thereto,e.g., at least 100, 150, or 200 or more contiguous amino acids.

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising at least one CDR (e.g., 1, 2, or 3) ora portion thereof from the amino acid sequence of SEQ ID NO:6 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a heavy chain variable region comprising atleast one CDR (e.g., 1, 2, or 3) or a portion thereof from an amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:5 or a sequenceat least 90% identical thereto, e.g., at least 95, 96, 97, 98, or 99%identical thereto. One of skill in the art understands that the CDRsplay an important role in binding specificity and that sequencesubstitutions (e.g., for humanization of a mouse antibody) arepreferably made outside of the CDRs and that minimal changes are madewithin the CDRs. Thus, in some embodiments, sequences that are at least90% identical to the disclosed sequences comprise no changes or only aminimal number of changes to the CDRs.

In one embodiment, the antibody or a fragment thereof comprises a lightchain variable region comprising at least one CDR (e.g., 1, 2, or 3) ora portion thereof from the amino acid sequence of SEQ ID NO:8 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a light chain variable region comprising atleast one CDR (e.g., 1, 2, or 3) or a portion thereof from an amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:7 or a sequenceat least 90% identical thereto, e.g., at least 95, 96, 97, 98, or 99%identical thereto.

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising at least one CDR (e.g., 1, 2, or 3)from the amino acid sequence of SEQ ID NO:6 or a sequence at least 90%identical thereto, e.g., at least 95, 96, 97, 98, or 99% identicalthereto, or encoded by the nucleotide sequence of SEQ ID NO:5 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto, and a light chain variable region comprisingat least one CDR (e.g., 1, 2, or 3) from the amino acid sequence of SEQID NO:8 or a sequence at least 90% identical thereto, e.g., at least 95,96, 97, 98, or 99% identical thereto, or encoded by the nucleotidesequence of SEQ ID NO:7 or a sequence at least 90% identical thereto,e.g., at least 95, 96, 97, 98, or 99% identical thereto.

As a further aspect, the invention provides compositions comprising theantibodies or fragments thereof of the invention. In some embodiments,the compositions are pharmaceutical formulations comprising theantibodies of the invention in a pharmaceutically acceptable carrier.

By “pharmaceutically acceptable” it is meant a material that is notbiologically or otherwise undesirable, i.e., the material can beadministered to a subject without causing any undesirable biologicaleffects such as toxicity.

The formulations of the invention can optionally comprise medicinalagents, pharmaceutical agents, carriers, adjuvants, dispersing agents,diluents, and the like.

The compounds of the invention can be formulated for administration in apharmaceutical carrier in accordance with known techniques. See, e.g.,Remington, The Science And Practice of Pharmacy (9^(th) Ed. 1995). Inthe manufacture of a pharmaceutical formulation according to theinvention, the compound (including the physiologically acceptable saltsthereof) is typically admixed with, inter alia, an acceptable carrier.The carrier can be a solid or a liquid, or both, and is preferablyformulated with the compound as a unit-dose formulation, for example, atablet, which can contain from 0.01 or 0.5% to 95% or 99% by weight ofthe compound. One or more compounds can be incorporated in theformulations of the invention, which can be prepared by any of thewell-known techniques of pharmacy.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g.,subcutaneous, intramuscular including skeletal muscle, cardiac muscle,diaphragm muscle and smooth muscle, intradermal, intravenous,intraperitoneal), topical (i.e., both skin and mucosal surfaces,including airway surfaces), intranasal, transdermal, intraarticular,intrathecal, and inhalation administration, administration to the liverby intraportal delivery, as well as direct organ injection (e.g., intothe liver, into the brain for delivery to the central nervous system,into the pancreas, or into a tumor or the tissue surrounding a tumor).The most suitable route in any given case will depend on the nature andseverity of the condition being treated and on the nature of theparticular compound which is being used.

For injection, the carrier will typically be a liquid, such as sterilepyrogen-free water, pyrogen-free phosphate-buffered saline solution,bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.). Forother methods of administration, the carrier can be either solid orliquid.

For oral administration, the compound can be administered in soliddosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Compounds can beencapsulated in gelatin capsules together with inactive ingredients andpowdered carriers, such as glucose, lactose, sucrose, mannitol, starch,cellulose or cellulose derivatives, magnesium stearate, stearic acid,sodium saccharin, talcum, magnesium carbonate and the like. Examples ofadditional inactive ingredients that can be added to provide desirablecolor, taste, stability, buffering capacity, dispersion or other knowndesirable features are red iron oxide, silica gel, sodium laurylsulfate, titanium dioxide, edible white ink and the like. Similardiluents can be used to make compressed tablets. Both tablets andcapsules can be manufactured as sustained release products to providefor continuous release of medication over a period of hours. Compressedtablets can be sugar coated or film coated to mask any unpleasant tasteand protect the tablet from the atmosphere, or enteric-coated forselective disintegration in the gastrointestinal tract. Liquid dosageforms for oral administration can contain coloring and flavoring toincrease patient acceptance.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the compound in a flavored base, usually sucrose andacacia or tragacanth; and pastilles comprising the compound in an inertbase such as gelatin and glycerin or sucrose and acacia.

Formulations suitable for parenteral administration comprise sterileaqueous and non-aqueous injection solutions of the compound, whichpreparations are preferably isotonic with the blood of the intendedrecipient. These preparations can contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the intended recipient. Aqueous and non-aqueous sterilesuspensions can include suspending agents and thickening agents. Theformulations can be presented in unit\dose or multi-dose containers, forexample sealed ampoules and vials, and can be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline or water-for-injection immediatelyprior to use.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a compound of theinvention, in a unit dosage form in a sealed container. The compound orsalt is provided in the form of a lyophilizate which is capable of beingreconstituted with a suitable pharmaceutically acceptable carrier toform a liquid composition suitable for injection thereof into a subject.The unit dosage form typically comprises from about 10 mg to about 10grams of the compound or salt. When the compound or salt issubstantially water-insoluble, a sufficient amount of emulsifying agentwhich is pharmaceutically acceptable can be employed in sufficientquantity to emulsify the compound or salt in an aqueous carrier. Onesuch useful emulsifying agent is phosphatidyl choline.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These can be prepared by admixing thecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which can be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration can be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration can also be delivered byiontophoresis (see, for example, Tyle, Pharm. Res. 3:318 (1986)) andtypically take the form of an optionally buffered aqueous solution ofthe compound. Suitable formulations comprise citrate or bis/tris buffer(pH 6) or ethanol/water and contain from 0.1 to 0.2M of the compound.

The compound can alternatively be formulated for nasal administration orotherwise administered to the lungs of a subject by any suitable means,e.g., administered by an aerosol suspension of respirable particlescomprising the compound, which the subject inhales. The respirableparticles can be liquid or solid. The term “aerosol” includes anygas-borne suspended phase, which is capable of being inhaled into thebronchioles or nasal passages. Specifically, aerosol includes agas-borne suspension of droplets, as can be produced in a metered doseinhaler or nebulizer, or in a mist sprayer. Aerosol also includes a drypowder composition suspended in air or other carrier gas, which can bedelivered by insufflation from an inhaler device, for example. SeeGanderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood(1987); Gonda (1990) Critical Reviews in Therapeutic Drug CarrierSystems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth.27:143 (1992). Aerosols of liquid particles comprising the compound canbe produced by any suitable means, such as with a pressure-drivenaerosol nebulizer or an ultrasonic nebulizer, as is known to those ofskill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solidparticles comprising the compound can likewise be produced with anysolid particulate medicament aerosol generator, by techniques known inthe pharmaceutical art.

Alternatively, one can administer the compound in a local rather thansystemic manner, for example, in a depot or sustained-releaseformulation.

A further aspect of the invention relates to kits for use in the methodsof the invention. The kit can comprise the antibody of the invention ina form suitable for administration to a subject or in a form suitablefor compounding into a formulation. The kit can further comprise othercomponents, such as therapeutic agents, carriers, buffers, containers,devices for administration, and the like. The kit can be designed fortherapeutic use, diagnostic use, and/or research use and the additionalcomponents can be those suitable for the intended use. The kit canfurther comprise labels and/or instructions, e.g., for treatment of adisorder. Such labeling and/or instructions can include, for example,information concerning the amount, frequency and method ofadministration of the antibody.

III. METHODS

As one aspect, the invention provides methods of depleting pDC in asubject, comprising delivering to the subject an effective amount of anantibody or a fragment thereof that specifically binds to BDCA2 anddepletes pDC, thereby depleting pDC. In some embodiments, pDC aredepleted by at least about 50% relative to subjects that have notreceived the antibody or fragment thereof, e.g., at least 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, or 99% or more. In other embodiments, theinvention provides methods of reducing the number of and/or depletingpDC in a sample ex vivo or in vitro, e.g., a mixed population of cells,comprising delivering to the sample an effective amount of an antibodyor a fragment thereof that specifically binds to BDCA2 and reduces thenumber of and/or depletes pDC, thereby reducing the number of and/ordepleting pDC.

In a further aspect, the invention provides methods of treating adisorder associated with pDC in a subject, comprising delivering to thesubject a therapeutically effective an antibody or a fragment thereofthat specifically binds to BDCA2 and reduces the number of and/ordepletes pDC, thereby treating the disorder.

In some embodiments, the subject is a research subject, e.g., alaboratory animal. In other embodiments, the subject is one that hasbeen diagnosed with a disorder associated with pDC. In anotherembodiment, the subject may be one that is at risk of developing adisorder associated with pDC (e.g., predisposed due to hereditaryfactors, exposure to a pathogen, abnormal immune cell counts, etc.).Disorders associated with pDC include, without limitation, infectiousdiseases or persistent virus infection (e.g., HIV infection), autoimmunedisease (e.g., systemic lupus erythematosus), and cancer (e.g.,pDC-derived leukemia), and disorders associated with tissue accumulationof pDC.

The antibodies of the present invention can optionally be delivered inconjunction with other therapeutic agents. The additional therapeuticagents can be delivered concurrently with the antibodies of theinvention. As used herein, the word “concurrently” means sufficientlyclose in time to produce a combined effect (that is, concurrently can besimultaneously, or it can be two or more events occurring within a shorttime period before or after each other).

In one embodiment, the antibodies of the invention are administered inconjunction with anti-cancer agents, such as 1) vinca alkaloids (e.g.,vinblastine, vincristine); 2) epipodophyllotoxins (e.g., etoposide andteniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D),daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin,plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes(e.g., L-asparaginase); 5) biological response modifiers (e.g.,interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatinand carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8)substituted ureas (e.g., hydroxyurea); 9) methylhydrazine derivatives(e.g., procarbazine (N-methylhydrazine; MIH)); 10) adrenocorticalsuppressants (e.g., mitotane (o,p′-DDD) and aminoglutethimide); 11)adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g.,hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrolacetate); 13) estrogens (e.g., diethylstilbestrol and ethinylestradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g.,testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g.,flutamide): and 17) gonadotropin-releasing hormone analogs (e.g.,leuprolide). In another embodiment, the compounds of the invention areadministered in conjunction with anti-angiogenesis agents, such asantibodies to VEGF (e.g., bevacizumab (AVASTIN), ranibizumab (LUCENTIS))and other promoters of angiogenesis (e.g., bFGF, angiopoietin-1),antibodies to alpha-v/beta-3 vascular integrin (e.g., VITAXIN),angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNF alphaconjugate, cyclophosphamide, combretastatin A4 phosphate,dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurin,paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation(Abraxane), soy isoflavone (Genistein), tamoxifen citrate, thalidomide,ADH-1 (EXHERIN), AG-013736, AMG-706, AZD2171, sorafenib tosylate,BMS-582664, CHIR-265, pazopanib, PI-88, vatalanib, everolimus, suramin,sunitinib malate, XL184, ZD6474, ATN-161, cilenigtide, and celecoxib.

in one embodiment, the antibodies of the invention are administered inconjunction with antiviral agents including, for example,virus-inactivating agents such as nonionic, anionic and cationicsurfactants, and C31 G (amine oxide and alkyl betaine), polybiguanides,docosanol, acylcarnitine analogs, octyl glycerol, and antimicrobialpeptides such as magainins, gramicidins, protegrins, and retrocyclins.Mild surfactants, e.g., sorbitan monolaurate, may advantageously be usedas antiviral agents in the compositions described herein. Otherantiviral agents that may advantageously be utilized in the compositionsdescribed herein include nucleotide or nucleoside analogs, such astenofovir, acyclovir, amantadine, didanosine, foscarnet, ganciclovir,ribavirin, vidarabine, zalcitabine, and zidovudine. Further antiviralagents that may be used include non-nucleoside reverse transcriptaseinhibitors, such as UC-781 (thiocarboxanilide), pyridinones, TIBO,nevaripine, delavirdine, calanolide A, capravirine and efavirenz. Fromthese reverse transcriptase inhibitors, agents and their analogs thathave shown poor oral bioavailability are especially suitable foradministration to mucosal tissue, in combination with antibodies andcompositions of the invention, to prevent sexual transmission of HIV.Other antiviral agents that may be used are those in the category of HIVentry blockers, such as cyanovirin-N, cyclodextrins, carregeenans,sulfated or sulfonated polymers, mandelic acid condensation polymers,monoclonal antibodies, chemokine receptor antagonists such as TAK-779,SCH-C/D, and AMD-3100, and fusion inhibitors such as T-20 and 1249.

In one embodiment, the antibodies of the invention are administered inconjunction with immunosuppressive agents including, for example,cyclosporine A, rapamycin, glucocorticoids, azathioprine, mizoribine,aspirin derivatives, hydroxychloroquine, methotrexate, cyclophosphamideand FK506 (tacrolimus).

In particular embodiments, the antibody is administered to the subjectin a therapeutically effective amount, as that term is defined above.Dosages of pharmaceutically active compounds can be determined bymethods known in the art, see, e.g., Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.). The therapeutically effectivedosage of the antibody will vary somewhat patient to patient, and willdepend upon the condition of the patient and the route of delivery. As ageneral proposition, a dosage from about 0.1 to about 50 mg/kg will havetherapeutic efficacy. Toxicity concerns at the higher level can restrictintravenous dosages to a lower level such as up to about 10 mg/kg. Adosage from about 10 mg/kg to about 50 mg/kg can be employed for oraladministration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg canbe employed for intramuscular injection.

In particular embodiments of the invention, more than one administration(e.g., two, three, four, or more administrations) can be employed over avariety of time intervals (e.g., hourly, daily, weekly, monthly, etc.)to achieve therapeutic effects.

The present invention finds use in veterinary and medical applicationsas well as research applications. As used herein, the term “subject”refers to humans and other animals. Suitable subjects include mammalssuch as humans, as well as those mammals of importance due to beingendangered, such as Siberian tigers; of economic importance, such asanimals raised on farms; animals of social importance to humans, such asanimals kept as pets or in zoos; and research animals, such as mice,rabbits, guinea pigs, ferrets, dogs, cats, monkeys, and apes. Examplesof such animals include but are not limited to: carnivores such as catsand dogs; swine, including pigs, hogs, and wild boars; ruminants and/orungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, andcamels; horses; and poultry.

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

Example 1 Experimental Methods

Construction of Humanized Mice:

Approval for animal work was obtained from the University of NorthCarolina Institutional Animal Care and Use Committee (IACUC). Weconstructed Balb/C rag2-gammaC (DKO) mutant DKO-hu HSC orNod-rag1-gammaC (NRG) NRG-hu HSC mice similarly as previouslyreported⁵⁰. Briefly, human CD34+ cells were isolated from 16- to20-week-old fetal liver tissues. Tissues were digested with Liver DigestMedium (Invitrogen, Frederick, Md.). The suspension was filtered througha 70 μm cell strainer (BD Falcon, Lincoln Park, N.J.) and wascentrifuged at 150 g for 5 minutes to isolate mononuclear cells byFicoll. After selection with the CD34+ magnetic-activated cell sorting(MACS) kit, CD34+ HSCs (0.5×10⁶) were injected into the liver of each 2-to 6-days old DKO or NRG mice, which had been previously irradiated at300 rad. More than 95% of the humanized mice were stably reconstitutedwith human leukocytes in the blood (10%-90% at 12-14 weeks). Each cohort(humanized mice reconstituted from the same human donor fetal livertissue) had similar levels of engraftment. All mice were housed at theUniversity of North Carolina at Chapel Hill.

HIV-1 Virus Stocks and Infection of Humanized Mice:

HIV-R3A, generated by cloning a highly pathogenic dual tropic envelopeinto NL4-3 backbone^(24,25,51), was used for acute infection experiment.An R5 tropic strain of HIV-1, JR-CSF, was used for both acute andchronic infection. All viruses were generated by transfection of 293Tcells. Humanized mice with stable human leukocyte reconstitution wereinfected with HIV-R3A or JR-CSF, at a dose of 10 ng p24/mouse, throughintravenous injection (i.v.). Humanized mice infected with 293T mocksupernatant were used as control groups.

Depletion of Human Plasmacytoid Dendritic Cells (pDC) in Humanized Mice:

A monoclonal antibody specific to blood dendritic cell antigen-2(BDCA2), 15B, was used to treat humanized mice through intraperitonealinjection (i.p., 4 mg/kg). For acute HIV-1 infection, humanized micewere injected three times with 15B on −5, −3 and −1 days beforeinfection. For chronic HIV-1 infection, 15B was applied to mice at 11weeks post-infection (wpi) by injecting twice every week for 10 weeks.

HIV-1 Viral Load Detection:

Viral genomic RNA in plasma was extracted using QIAamp® Viral RNA MiniKit (QIAGEN, cat#52904) according to the manufacture's instruction.HIV-1 replication (genome copy/ml in the plasma) was measured byReal-Time PCR (ABI Applied Biosystem).

Animal Termination and Tissue Processing:

For acute HIV-1 infection, mice were terminated at 1 wpi (NL4-R3A) or 3wpi (JR-CSF). For chronic JR-CSF infection, mice were terminated at 21wpi. On termination, total leukocytes were isolated from mouse lymphoidorgans as previously described^(50,52). Lymphoid tissues, includingperipheral blood (PBL), mesenteric lymph nodes (mLN), spleen (SP) andbone marrow (BM) were harvested for analysis. Red blood cells were lysedwith ACK buffer, and the remaining cells were stained and fixed with 1%(wt/vol) formaldehyde before FACS analysis. Total cell number wasquantified by Guava Easycytes with Guava Express software (Guava).

Flow Cytometry:

For HIV-1 gag p24 staining, cells were stained with surface antibodiesfirst, then permeabilized with cytofix/cytoperm buffer (BD Bioscience,cat#554714), followed by intracellular staining. Human leukocytes(mCD45-huCD45+) were analyzed for human CD3, CD4, CD8, CD123, HLA-DR andCD38 by CyAn FACS machine (Dako). FITC-conjugated anti-human HLA-DR(clone:L243, cat#307604), PE-conjugated anti-human CD38 (clone:HIT2,cat#303506), PE/Cy5-conjugated anti-human CD4 (clone:RP4-T4,cat#300510), PE/Cy7-conjugated anti-human CD3 (clone:HIT3a, cat#300316),Pacific blue-conjugated anti-human CD3 (clone:UCHT1, cat#300431),PE/Cy7-conjugated anti-human CD8 (clone:HIT8a, cat#300914),APC-conjugated human CD123 (clone:6H6, cat#306012) and APC/Cy7-conjuagedanti-human CD45 (clone:H130, cat#304014) were purchased from Biolegend;PE-conjugated anti-human caspase-3 (clone:C92-605, cat#51-68655X) waspurchased from BD Bioscience. Pacific orange-conjugated anti-mouse CD45(clone:H130, cat#MHCD4530), PE/Texas red-conjugated anti-human CD4(clone:S3.5, cat#MHCDO417) or CD8 (clone:3B5, cat#MHCD0817), andLIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (cat#L34957) were purchasedfrom Invitrogen. FITC-conjugated anti-HIV p24 (clone:FH190-1-1,cat#6604665) was purchased from Beckman Coulter. The cells were analyzedon a CyAn ADP (Dako).

Human Cytokine Luminex Assay:

Cytokines in the mouse plasma were quantified with Milliplex®MAP umanCytokine/Chemokine Magnetic Bead Panel Immunoassay (Millipore). Plasmasamples were collected and stored at −80° C. before analysis. The assayswere performed at Clinical Proteomics Laboratory at University of NorthCarolina at Chapel Hill.

Immunohistochemistry:

Paraffin-embedded spleen sections from humanized mice were stained withthe mouse anti-human huCD45 antibody (Dako, cat#N1514), washed in PBS,then incubated with Mouse-&-Rabbit-on-Rodent Double Stain Polymer(BIOCARE MEDICAL, cat#RDS513H) and substrate DAB (BIOCARE MEDICAL,cat#BDB2004 H, L, MM). Images were captured using a QImagingMicropublisher 3.3 CCD digital camera and QCapture software version 3.0(QImaging, Surrey, BC).

Cell Purification by FACS Sorting:

Spleen cells from mock or treated groups of mice were pooled. For humanCD45+ cells sorting, total m spleen were stained with human CD45, mouseCD45 and 7-Aminoactinomycin D (7-AAD). For human CD8+ T cell sorting,CD3 and CD8 antibody were added to the antibody mix. Cell sorting wasperformed by the UNC Flow Cytometry Core.

Agilent Microarray Assay:

RNA purification was done using RNeasy® Plus Mini Kit (QIAGEN,cat#74134) according to the manufacture's instruction. DNase-RNase free(QIAGEN) treatment was added to the column to eliminate any potentialDNA contamination during RNA preparations. Total RNA was checked forquantity, purity and integrity by capillary electrophoresis. RNA wasamplified with Cy3- and Cy5-labeled CTP in separate reactions to producedifferentially labeled samples and reference c-DNAs. 200 to 400 ng oftotal RNA were used as a starting material to prepare cDNA. Both werehybridized to the same microarray (UNC Genomic and Bioinformatics Core)using SurePrint G3 Human Gene Expression 8×60K Microarray Kit (Agilent).Agilent Feature Extraction v18 software was used to analyze all images.Gene expression values were quantified by the log 2 ratio of red channelintensity (mean) vs. green channel intensity (mean), followed by LOWESSnormalization to remove the intensity dependent dye bias⁵³.

Cellular mRNA Level Detection:

Interferon alpha-1/13 (IFNα1/13), interferon alpha-2 (IFNα2), interferonbeta (IFNβ)⁵⁴, interferon gamma (IFNγ)⁵⁵ and tumor necrosis factor alpha(TNFα)⁵⁶ were detected. Type I interferon stimulated genes, M×A⁵⁷ andTRLM22⁵⁸, were detected to confirm pDCs depletion effect on type I IFNproduction. Real-time PCR assay was performed (ABI Applied Biosystem).All samples were tested in triplicate using the human GAPDH gene⁵⁹ fordata normalization.

Statistical Analysis:

Data were analyzed using GraphPad Prism software version 5.0 (GraphPadsoftware, San Diego, Calif., USA). The methods used for analysis ofmicroarray data were described above. The data from different cohorts ofmice were compared using a 2-tailed Mann-Whitney U test. For geneexpression, mean-ΔCT was calculated as the average (±SD) of all ΔCTvalues within each group of samples and 2-way ANOVA method was used.Correlations were estimated with a Spearman test. All results wereconsidered significant when p<0.05.

Example 2 HIV-1 Infection in Humanized Mice

Others and we have reported that functional human pDC are developed inlymphoid tissues in humanized mouse models (Traggiai et al., Science304:104 (2004); Zhang et al., Blood 117:6184 (2011); Tanaka et al., J.Immunol. (2012)). Human pDC are rapidly activated by HIV-1 infection andthe level of pDC activation is reversely correlated with CD4⁺ T-cellnumbers (Zhang et al., Blood 117:6184 (2011)), which is consistent withthe observation from HIV-1 infected patients (Buimovici-Klein et al.,Lancet 2:344 (1983); Buimovici-Klein et al., AIDS Res. 2:99-108 (1986);Meier et al., Nature Medicine 15:955 (2009)) and SIV infected monkeys(Harris et al., J. Virol. 84:7886 (2010); Campillo-Gimenez et al., J.Virol. 84:1838 (2010)). In this study it was shown that persistent HIVinfection is efficiently established in humanized mice infected witheither the dual-tropic R3A strain (FIG. 1a ) or CCR5-tropic JR-CSFstrain (FIG. 2). In both acute and chronic HIV-1 infection, an increaseof HLA-DR⁺CD38⁺ CD8 T cells was observed (FIG. 1b , FIGS. 3a-3c ), alongwith a decrease of CD4 T cell percentage in CD3⁺ T cell (FIG. 1e , FIGS.4a-4c ). As in HIV-1 patients, leukocytopenia, or depletion of totalhuman CD45 leukocytes including CD8 T cells, also occurred in the bloodand spleen (FIG. 1d , FIG. 5 a). Similar as in HIV patients, there was asignificant induction of type I interferon production in plasma ineither R3A or JR-CSF infected mice (FIG. 1e , FIG. 5b ), accompaniedwith an increase of type I interferon specific ISG genes expression inleukocytes from spleen (FIG. 1f , FIG. 5c ). Thus, humanized miceprovide a relevant in vivo model for studying the role of HIV-1 and hostimmune effectors in HIV-1 immunopathogenesis (Zhang et al., Blood117:6184 (2011); Zhang et al., Cell. Mol. Immunol. 9:237 (2012)).

Example 3 Depletion of Plasmacytoid Dendritic Cells in Humanized Mice

In order to delineate the role of human pDC in HIV-1 infection andpathogenesis in vivo, an anti-BDCA2 (CD303) monoclonal antibody (15B)was developed, which could specifically deplete human pDC in lymphoidorgans of humanized mice. After 15B injection, human pDC in CD45leukocytes was greatly reduced in both peripheral blood (FIG. 6a ) andlymphoid organs (FIG. 6b ). As controls, human T, B,monocytes/macrophages and myeloid dendritic cells were not perturbed by15B injection (FIGS. 7-9).

Example 4 Role of Plasmacytoid Dendritic Cells in Acute HIV-1 Infection

To test the roles of pDC during early primary HIV-1 infection, 15B andisotype control antibodies were injected into humanized mice on −5, −3and −1 days before infection, and the mice infected with R3A, a highlypathogenic, CCR5− and CXCR4-dual tropic HIV-1 strain (Meissner et al.,Virology 328:74 (2004); Sivaraman et al., J. Virol. 83:11715 (2009)).The infected mice were injected with 15B or control antibody on 3 and 6days post-infection. It was found that pDC remained depleted in bloodand lymphoid organs of the infected mice (FIG. 10a ), when terminated on8 days post-infection. Interestingly, the plasma IFN-I level wassignificantly abolished by pDC depletion in HIV-1 infected mice (FIG.10b ). The induction of different subtypes of human IFN-I was alsoabolished at RNA level by real time PCR (FIG. 10c ). In addition, theupregulation of ISGs such as Mx1, TRIM22 was also blocked (FIG. 10d ).These data demonstrate that pDCs are the major, if not the only, IFN-1producing cells in vivo during early HIV-1 infection in humanized mice.

Consistent with the antiviral activity of IFN-I, pDC depletion led toelevated HIV-1 replication in vivo. The average plasma viremia wasincreased about 10-fold (p<0.01) comparing with control antibody treatedmice (FIG. 11a ). The experiment was repeated with the less pathogenic,CCR5 tropic HIV-1 JR-CSF. Similar to R3A infection, JR-CSF replicationwas also increased about 5-fold in pDC-depleted mice (FIG. 11c ). Theincrease of viral replication was further confirmed byimmunohistochemistry (FIG. 11b ) or flow cytometry (FIGS. 11d,e ) forHIV p24 protein positive cells in human cells from spleens.

HIV-1 infection induces generalized immune activation (Lane et al., N.Engl. J. Med. 309:453 (1983); Ascher et al., Clin. Exp. Immunol. 73:165(1988)), which is proposed to contribute to HIV-1 diseases progression(Lane et al., N. Engl. J. Med. 309:453 (1983); Ascher et al, Clin. Exp.Immunol. 73:165 (1988); Grossman et al., Clin. Immunol. Immunopathol.69:123 (1993); Giorgi et al., J. Acquir. Immune Defic. Syndr. 6:904(1993); Bosinger et al., Curr. Opin. HIV AIDS 6:411 (2011); Moir et al.,Annu. Rev. Pathol. 6:223 (2011)). Induction of IFN-I and pDC activationhave been hypothesized to contribute to the immune activation both inHIV-infected patients and in SIV-infected rhesus monkeys(Buimovici-Klein et al., Lancet 2:344 (1983); Buimovici-Klein et al.,AIDS Res. 2:99-108 (1986); Meier et al., Nature Medicine 15:955 (2009);Harris et al., J. Virol. 84:7886 (2010); Campillo-Gimenez et al., J.Virol. 84:1838 (2010); Bosinger et al., Curr. Opin. HIV AIDS 6:411(2011); Kwa et al., Blood 118:2763 (2011); Manches et al., Proc. Natl.Acad. Sci. USA 109:14122 (2012); Manches et al., J. Clin. Invest.118:3431 (2008)). However, instead of decreasing T cell activation, afurther increase of T cell activation (CD38+DR+) was observed in bothblood and lymphoid organs in pDC-depleted mice infected with either R3A(FIGS. 12a,b ) or JR-CSF (FIGS. 12c,d ). Interestingly, it was foundthat the CD8 T cell activation level was correlated with viral load inpDC-depleted mice (FIG. 13). Thus, HIV-1 may also directly lead to theT-cell activation in the absence of INF-I.

However, despite the increased HIV-R3A viral replication, the absolutenumbers of human CD4⁺ T cell in blood and spleen were comparable tothose of control antibody treated mice (FIG. 14a ). More surprisingly,CD8⁺ cells of pDC depleted mice increased significantly compared withcontrol antibody treated animals (FIG. 14b ). Total human CD45⁺leukocytes were also preserved in blood and spleen (FIG. 14c ), and thisis correlated with decreased cell death of total CD45+ or CD8 T cells(FIGS. 14d,e ). Similar findings were observed in JR-CSF infected mice,although only low levels of human CD4⁺, CD8+ T cells and total humanleukocytes and depletion occurred at 3 weeks post-infection (FIG. 15).

EXAMPLE 5 Role of Plasmacytoid Dendritic Cells in Chronic HIV-1Infection

pDC depletion was performed in humanized mice with establishedpersistent HIV-1 infection. Humanized mice were infected with JR-CSF for11 weeks; 15B was then applied to deplete pDCs. In agreement with thedata from pre-infection pDC depletion, an increased viremia was observedthat persisted for 10 weeks until termination (FIG. 16a ). Thepercentage of HIV infected cells (HIV-1 p24 positive) also increased(FIG. 16b , FIG. 17a ). Induction of plasma IFN-α2 was decreasedsignificantly in the pDC-depleted mice (FIG. 17b ). The diminishedinduction of different IFN-1 subtypes (FIG. 17c ) and ISGs (FIG. 17d )at RNA level in human leukocytes in spleen were also evidenced byreal-time PCR assay and cDNA array (FIGS. 17e,f ). These data suggestthat pDC are still the major INF-I producing cells during HIV-1 chronicinfection in vivo.

In spite of the persistent higher viremia during those 10 weeks of pDCdepletion, human CD4⁺ T-cell numbers increased significantly in thespleen, comparing to the control group (FIG. 16c , p<0.05). In addition,human CD8 T cell and CD45⁺ leukocyte numbers were also increased (FIGS.16d,e , p<0.01). Interestingly, human CD4, CD8 T cells and total CD45+leukocytes were not significantly rescued in the blood (FIGS. 16c-e ).The increase of human CD45+ cells was also evidenced by CD45immunohistochemistry stain of spleen sections (FIG. 16f ). Accordingly,pDC depletion significantly reduced the percentage of dead/dying cellsin T cells and total human CD45⁺ cells in the spleen (FIG. 16g ).Therefore, during chronic HIV-1 infection, pDC still play both a role insuppressing viral replication and enhancing HIV-inducedimmunopathogenesis.

The role of pDC in chronic virus infections such as HIV-1 remainscontroversial, due to the correlative studies performed in humanpatients or in SIV-infected monkeys (Cervantes-Barragan et al., Proc.Natl. Acad. Sci. USA 109:3012 (2012); Riviere et al., J. Exp. Med.152:633 (1980); Wang et al., Cell Host Microbe 11:631 (2012)). Here itis reported that, by depleting pDC specifically with a novel antibodybefore or during HIV-1 infection, pDC are the major IFN-I producingcells during HIV infection in vivo. It is reported that pDC play a dualrole during HIV-1 infection and pathogenesis: they produce IFN-I toinhibit HIV-1 replication, but enhance HIV-1 pathogenesis by promotingdeath of human leukocytes including human CD4 and CD8 T cells. Theresidual IFN-I expression after 15B mAb treatment during persistentHIV-1 infection may be due to residual pDC in the bone marrow, althoughthe contribution of other cells types cannot be excluded (Lepelley etal., PLoS Pathogens 7:e1001284 (2011)).

It is reported that pDC may contribute to HIV induced immune activationand subsequent immunopathogenesis. The anti-malaria drug chloroquineinhibits IFN-1 production by pDC In vitro (Beignon et at, J Clin.Invest. 115:3265 (2005)) and seems to rescue human T cells in HIV-1infected patients, correlated with reduced immune activation (Murray etal. J. Virol. 84:12082 (2010); Piconi et al., Blood 118:3263 (2011)).Interestingly, HIV-activated pDCs also induce regulatory T cells (Tregs)through an indoleamine 2,3-dioxygenase (IDO)-dependent mechanism(Manches et al., Proc. Natl. Acad. Sci. USA 109:14122 (2012); Manches etal., J. Clin. Invest. 118:3431 (2008)). In this report, an increase ofT-cell activation by pDC depletion was observed, probably due to theincreased viral replication via a pDC/IFN-I independent mechanism. Arecent report shows that a TLR7 and TLR9 antagonist that could inhibitactivation of pDC isolated from Rhesus monkeys did not significantlychange plasma IFN-I level and ISG expression in SIV infected monkeys,probably due to its incomplete inhibition of pDC in vive or to the highlevel activation of mDC and macrophages (Kader et al., PLoS Pathogens9:e1003530 (2013)). Thus, the relative roles of pDC and HIV-1replication, as well as other immune cells, in immune activation need tobe further investigated.

Repeated administrations of TLR7 ligands in mice induce AIDS-likelymphopenia, with reduced CD4⁺ T cells, CD8⁺ T cells and B cells(Baenziger et al., Blood 113:377 (2009)). It is reported that IFN-1triggers proapoptotic and antiproliferative effect on T cells (Tanabe etal., J. Immunol. 174:609 (2005)), and activation of Stat4 by TCRsignaling could overcome its STAT1-dependent inhibition of T cellsproliferation (Gil et al., Blood 120:3718 (2012)). Similarly, TLR7 andTLR9 antagonist DV056-treated macaques show a significant increase inproliferating memory CD4⁺ and CD8⁺ T cells in blood (Kader et al., PLoSPathogens 9:e1003530 (2013)). Consistently, it was found that pDCdepletion not only rescued CD4⁺ T cells but also total CD45⁺ leukocytesand CD8 T cells. It is proposed that pDC may contribute to HIVimmunopathogenesis by both inducing abnormal immune activation and bypromoting depletion of human immune cells.

Recent reports also show that blocking IFN-I signaling during LCMVpersistent infection could improve antiviral T cell response andaccelerate clearance of chronic LCMV infection via an IL-10-dependentmechanisms (Wilson et al., Science 340:202 (2013); Teijaro et al.,Science 340:207 (2013)). Similar findings were seen in our HIV-1infection model, as the enhanced levels of CD8 T cells and IFNγ werecorrelated with pDC depletion and blocking of IFN-I induction. However,pDC-depletion did not seem to affect IL-10 expression in humanized mice,likely due to the elevated levels of HIV-1 replication. In HAART-treatedHIV patients, a significant fraction fail to show reduced immuneactivation and efficient immune reconstitution even with efficientvirological responses (Aiuti et al., AIDS Rev. 8:88 (2006); Gaardbo etal., Clin. Dev. Immunol. 2012:670957 (2012); Zhang et al., Aids 27:1283(2013)). Persistent pDC activation and IFN-I induction may play a rolein such immune no-responder patients. It is proposed that inhibition ordepletion of pDC during HAART in HIV-1 chronic infection may provide aneffective treatment to preserve human immune cells in HIV-1 infectedpatients.

Example 6 Depletion of Plasmacytoid Dendritic Cells in Humanized Mice

Further screening of anti-BDCA2 (CD303) monoclonal antibodies identifiedanother antibody (12B) that specifically depletes human pDC in lymphoidorgans of humanized mice. After 12B injection, human pDC in CD45⁺leukocytes was greatly reduced in spleen (FIG. 20). As controls, human Tand myeloid dendritic cells were not perturbed by 12B injection (FIG.20).

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-19. (canceled)
 20. An antibody or a fragment thereof that specificallybinds to BDCA2 and depletes pDC when administered to a subject.
 21. Theantibody or a fragment thereof of claim 20 which is a monoclonalantibody or a fragment or derivative thereof.
 22. The antibody or afragment thereof of claim 21, which specifically binds the epitopeIQNLKRNSSYFLGLSDPGGR (SEQ ID NO:9) or a fragment thereof of at least 5contiguous amino acids.
 23. A monoclonal antibody or a fragment thereofcomprising a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:2 or a sequence at least 90% identical thereto,and/or a light chain variable region comprising the amino acid sequenceof SEQ ID NO:4 or sequence at least 90% identical thereto. 24.(canceled)
 25. A monoclonal antibody or a fragment thereof comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:6 or a sequence at least 90% identical thereto, and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO:8 or asequence at least 90% identical thereto.
 26. (canceled)
 27. Themonoclonal antibody or a fragment thereof of claim 23, which is achimeric antibody or a fragment thereof.
 28. The monoclonal antibody ora fragment thereof of claim 23, which is a humanized antibody or afragment thereof.
 29. A pharmaceutical composition comprising themonoclonal antibody or a fragment thereof claim 23 and apharmaceutically acceptable carrier.
 30. A kit comprising the monoclonalantibody or a fragment thereof of any one of claim 23 and instructionsfor use.
 31. The monoclonal antibody or a fragment thereof of claim 25,which is a chimeric antibody or a fragment thereof.
 32. The monoclonalantibody or a fragment thereof of claim 25, which is a humanizedantibody or a fragment thereof.
 33. A pharmaceutical compositioncomprising the monoclonal antibody or a fragment thereof claim 25 and apharmaceutically acceptable carrier.
 34. A kit comprising the monoclonalantibody or a fragment thereof of any one of claim 25 and instructionsfor use.